PROJECT SUMMARY/ABSTRACT Modifying T cells with chimeric antigen receptors (CARs) is a clinically-validated approach for the treatment of some B-cell malignancies. Though efficacious, the genetic modification of these cells is associated with significant drawbacks. For instance, the genetic engineering techniques are tedious, inefficient, and not applicable to all cell types. Furthermore, the modifications are permanent, leading to persistent, severe, and irreversible patient side effects. Because of these drawbacks, the use of an alternative, reversible scaffold to direct therapeutic cell-cell interactions would be highly beneficial. It has been previously demonstrated that a fusion protein comprised of two units of E. coli dihydrofolate reductase (DHFR2) will spontaneously assemble into a chemically self-assembled nanoring (CSAN) when combined with the chemical dimerizer bis- methotrexate (bisMTX). Recently, an anti-EpCAM scFv was fused to the DHFR2 protein, and a phospholipid was conjugated to the bisMTX species. Assembly of these species formed chemically self-assembled chimeric antigen receptors (CS-CARs) that were embedded in the membrane of T cells and drove selective recognition and killing of EpCAM-positive carcinoma cells. Importantly, the CS-CARs were readily removed from the T cell surface via incubation with the FDA-approved antibiotic trimethoprim, affording a pharmacological mechanism to deactivate the CS-CARs. Despite these positive in vitro results, it remains unclear whether the current structure of the CS-CAR is optimal for the initiation and maintenance of cell-cell interactions. Therefore, the aims proposed in this project are designed to further test the hypothesis that CS-CARs can be used as a rapid, reversible method to modify cell surfaces for therapeutic purposes. Specifically, the results will further the understanding of the structural components of these CS-CARs, providing a rational pathway to optimize their therapeutic efficacy. Aim 1 will focus on a systematic evaluation of each component of the CS-CAR, generating a small library of CS-CAR constructs. This includes variation of lipid species, PEG/peptide linker lengths, and targeting element identity (scFv vs. novel fibronectin engineered via yeast surface display). The ability of the CS-CARs to initiate and maintain specific, reversible cell-cell interactions in vitro will be also established. Aim 2 will evaluate the in vitro efficacy T cells modified with different anti-EpCAM CS-CARs to selectively recognize and kill EpCAM-positive MCF7 breast adenocarcinoma cells. These results will afford optimized CS-CARs suitable for further evaluation in vivo and enhance the field's understanding of designing reversible, therapeutic cell-cell interactions. Therefore, this proposal has broad implications not only for the cell-based treatment of malignancies, but also for other applications utilizing directed cell-cell interactions. Moreover, this application provides a rigorous, yet defined scientific and mentoring framework to foster the applicant's goals of becoming a successful academic physician-scientist.